本申请是申请日为2020年2月21日、申请号为202010109153.5、发明名称为“金诺芬等老药及其组合物在抗单正链RNA病毒中的应用”的发明专利申请的分案申请。This application is a divisional application of the invention patent application with the application date of February 21, 2020, application number 202010109153.5, and invention name "Application of old drugs such as Auranofin and their compositions in anti-single positive-strand RNA virus".
技术领域Technical Field
本发明涉及医药领域，具体地涉及金诺芬等多个老药及其组合物在抗单正链RNA病毒中的应用。The present invention relates to the field of medicine, and in particular to the application of a plurality of old drugs such as auranofin and their compositions in resisting single positive-strand RNA viruses.
背景技术Background Art
在急性传染病中，绝大部分都是病毒性传染病，病毒性传染病的发病率高，死亡率也很高。由于检测和诊断手段有限，导致新病毒引发的新疫情爆发往往具有突发性、随机性和不可预测性等特点，一旦爆发，如无有效的防治手段，极易造成大规模流行，严重威胁人民健康生命安全。Among acute infectious diseases, the vast majority are viral infectious diseases, which have high morbidity and mortality rates. Due to limited detection and diagnosis methods, new outbreaks caused by new viruses are often sudden, random, and unpredictable. Once an outbreak occurs, if there is no effective prevention and control method, it is very easy to cause a large-scale epidemic, seriously threatening people's health and life safety.
SARS-CoV-2病毒传播途径未完全掌握，已知能通过飞沫和接触传播，且存在人传人、医务人员感染，一定社区传播风险，且病毒存在变异的可能。目前对于新型冠状病毒所致疾病没有特异的预防和治疗方法。The transmission route of SARS-CoV-2 is not fully understood. It is known that it can be transmitted through droplets and contact, and there is a risk of human-to-human transmission, medical staff infection, and certain community transmission. The virus may mutate. Currently, there is no specific prevention or treatment for diseases caused by the new coronavirus.
SARS-CoV-2冠状病毒属于冠状病毒科冠状病毒属，为具有包膜的单链正义RNA病毒。和其他已知冠状病毒类似，SARS-CoV-2冠状病毒也经过吸附、穿入、脱壳、生物合成、子代病毒的组装与释放等几个过程完成子代病毒的增殖。SARS-CoV-2冠状病毒感染宿主细胞起始于病毒包膜表面的刺突糖蛋白与宿主细胞表面的ACE2受体结合，随后发生膜融合，病毒进入宿主细胞，在细胞溶酶体等细胞器作用下，释放出病毒的遗传物质单链正义RNA，在宿主细胞的线粒体、核糖体等蛋白质合成元件以及必须的原料等作用下，翻译产生多聚蛋白，之后，SARS-CoV-2冠状病毒的两大必需半胱氨酸蛋白酶：木瓜样蛋白酶(papain-likeprotease,PLpro)和3C样蛋白酶(3C-like protease,3CLpro)在特定位点切割加工多聚蛋白前体，产生多个对病毒生命周期非常重要的非结构蛋白。在这些非结构蛋白的作用下，病毒RNA复制出子代病毒核酸物质，并大量翻译出所需的结构蛋白，完成子代病毒的组装和释放。SARS-CoV-2冠状病毒感染细胞的生命周期的任何环节或关键酶均可以作为抗病毒药物的研究靶点，如水解切割多聚蛋白前体的半胱氨酸蛋白酶PLpro和3CLpro，负责完成子代病毒遗传物质复制的RNA聚合酶等。SARS-CoV-2 coronavirus belongs to the genus Coronavirus of the family Coronavirus, and is a single-stranded positive RNA virus with an envelope. Similar to other known coronaviruses, SARS-CoV-2 coronavirus also completes the proliferation of progeny viruses through several processes such as adsorption, penetration, uncoating, biosynthesis, assembly and release of progeny viruses. SARS-CoV-2 coronavirus infection of host cells begins with the binding of the spike glycoprotein on the surface of the viral envelope to the ACE2 receptor on the surface of the host cell, followed by membrane fusion, the virus enters the host cell, and under the action of organelles such as lysosomes, the virus's genetic material single-stranded positive RNA is released. Under the action of protein synthesis elements such as mitochondria and ribosomes of the host cell and necessary raw materials, it is translated to produce polyproteins. Afterwards, the two essential cysteine proteases of SARS-CoV-2 coronavirus: papain-like protease (PLpro ) and 3C-like protease (3CLpro ) cut and process polyprotein precursors at specific sites to produce multiple non-structural proteins that are very important for the life cycle of the virus. Under the action of these non-structural proteins, viral RNA replicates progeny viral nucleic acid materials and translates the required structural proteins in large quantities to complete the assembly and release of progeny viruses. Any link or key enzyme in the life cycle of SARS-CoV-2 coronavirus infected cells can be used as a research target for antiviral drugs, such as cysteine proteases PLpro and 3CLpro that hydrolyze and cleave polyprotein precursors, and RNA polymerases that are responsible for completing the replication of progeny viral genetic material.
3CL蛋白酶(3chymotrypsin-like protease,3CLpro)，又称主蛋白酶(Mpro)，是冠状病毒RNA翻译出多聚蛋白pp1a和pp1ab后水解产生多个非结构蛋白过程中的关键蛋白酶，对病毒的复制和感染至关重要，抑制3CL蛋白酶的催化功能可有效抑制病毒多聚蛋白前体的切割，阻断病毒复制，抑制子代病毒生成。3CLpro属于半胱氨酸蛋白酶，在所有冠状病毒中的序列和结构高度保守，与小RNA病毒中的3C蛋白酶也非常相似，而且人体中没有与其相似的蛋白酶。因此，3CLpro是目前公认的研发广谱抗单正链RNA病毒药物的理想靶点。3CL protease (3chymotrypsin-like protease, 3CLpro ), also known as main protease (Mpro ), is a key protease in the process of hydrolyzing multiple non-structural proteins after coronavirus RNA translates polyproteins pp1a and pp1ab. It is essential for viral replication and infection. Inhibiting the catalytic function of 3CL protease can effectively inhibit the cleavage of viral polyprotein precursors, block viral replication, and inhibit the generation of progeny viruses. 3CLpro is a cysteine protease. Its sequence and structure are highly conserved in all coronaviruses. It is also very similar to the 3C protease in small RNA viruses, and there is no similar protease in the human body. Therefore, 3CLpro is currently recognized as an ideal target for the development of broad-spectrum anti-single positive-strand RNA virus drugs.
目前，针对SARS-CoV-2冠状病毒导致的严重疾病(COVID-19)尚无特效的抗病毒药物。这些感染性疾病严重影响了人们的生命健康，研发效果好的抗病毒药物迫在眉睫。针对SARS-CoV-2冠状病毒3CLpro开发出低毒高效的抗病毒药物，以满足国内外SARS-CoV-2冠状病毒感染患者的临床需求，具有重大的社会意义。At present, there is no specific antiviral drug for severe diseases (COVID-19) caused by SARS-CoV-2 coronavirus. These infectious diseases have seriously affected people's lives and health, and the development of effective antiviral drugs is urgent. The development of low-toxic and highly effective antiviral drugs for SARS-CoV-2 coronavirus 3CLpro to meet the clinical needs of SARS-CoV-2 coronavirus infected patients at home and abroad has great social significance.
综上所述，本领域迫切需要开发针对SARS-CoV-2冠状病毒3CL蛋白酶的抑制剂用于治疗新型冠状病毒感染引起的肺炎及其他单正链RNA病毒感染。In summary, there is an urgent need in the art to develop inhibitors against SARS-CoV-2 coronavirus 3CL protease for the treatment of pneumonia caused by the new coronavirus infection and other single positive-strand RNA virus infections.
发明内容Summary of the invention
本发明的目的是提供可有效抑制冠状病毒3CL蛋白酶的药物分子及其在抑制单正链RNA病毒感染方面的新用途。The purpose of the present invention is to provide a drug molecule that can effectively inhibit coronavirus 3CL protease and a new use thereof in inhibiting single positive-strand RNA virus infection.
具体地，本发明提供了金诺芬等多个老药及其组合物在抗单正链RNA病毒感染中尤其是新型冠状病毒疾病(COVID-19)中的用途。Specifically, the present invention provides the use of multiple old drugs such as Auranofin and their compositions in combating single positive-strand RNA virus infection, especially the new coronavirus disease (COVID-19).
在本发明第一方面，提供了一种活性成分或含所述活性成分的制剂的用途(或制药用途)，所述的活性成分选自下组：In a first aspect of the present invention, there is provided a use (or pharmaceutical use) of an active ingredient or a preparation containing the active ingredient, wherein the active ingredient is selected from the following group:
(Z1)选自A1～A73中任一活性成分(老药)、或其药学上可接受的盐或其提取物；(Z1) any active ingredient selected from A1 to A73 (old drug), or a pharmaceutically acceptable salt or extract thereof;
(A1)金诺芬；(A1) Auranofin;
(A2)雷贝拉唑钠；(A2) rabeprazole sodium;
(A3)肝素钠；(A3) Heparin sodium;
(A4)双硫仑；(A4) Disulfiram;
(A5)艾普拉唑；(A5) ilaprazole;
(A6)博赛泼维；(A6) Boceprevir;
(A7)泰妥拉唑；(A7) Tenatoprazole;
(A8)依沙吖啶；(A8) ethacridine;
(A9)依沙吖啶；(A9) ethacridine;
(A10)布罗波尔；(A10) Brobol;
(A11)磺达肝素钠；(A11) Fondaparinux sodium;
(A12)硫糖铝；(A12) sucralfate;
(A13)辛烯啶；(A13) octenidine;
(A14)六氯酚；(A14) Hexachlorophene;
(A15)伊文思蓝；(A15) Evans blue;
(A16)去甲二氢化愈创木酸；(A16) nordihydroguaiaretic acid;
(A17)溴化钍；(A17) Thorium bromide;
(A18)单宁酸；(A18) tannic acid;
(A19)奥美拉唑；(A19) omeprazole;
(A20)苄塞拉齐特(塞拉齐特)；(A20) benzylazepide (celazide);
(A21)非洛地平；(A21) felodipine;
(A22)利匹韦林；(A22) Rilpivirine;
(A23)苯氧基苯胺；(A23) phenoxyaniline;
(A24)蒽林；(A24) anthralin;
(A25)二巯基丁二酸；(A25) dimercaptosuccinic acid;
(A26)左旋甲状腺素钠；(A26) Levothyroxine sodium;
(A27)头孢克洛；(A27) Cefaclor;
(A28)替拉曲考；(A28) Tilatracol;
(A29)奎宁；(A29) Quinine;
(A30)TIBSOVO；(A30)TIBSOVO;
(A31)碘塞罗宁；(A31) liothyronine;
(A32)苯溴马隆；(A32) Benzbromarone;
(A33)奎宁；(A33) Quinine;
(A34)双碘喹啉；(A34) iodoquinol;
(A35)格拉司琼；(A35) Granisetron;
(A36)米安色林；(A36) Mianserin;
(A37)卡格列净；(A37) Canagliflozin;
(A38)贝达喹啉；(A38) Bedaquiline;
(A39)三氯生；(A39) triclosan;
(A40)齐拉西酮；(A40) Ziprasidone;
(A41)艾尔巴韦；(A41) Elbavir;
(A42)贝达喹啉；(A42) Bedaquiline;
(A43)五氯柳胺；(A43) pentachlorosulamide;
(A44)氯化十六烷吡啶；(A44) hexadecylpyridinium chloride;
(A45)非诺多泮；(A45) fenoldopam;
(A46)三碘甲状腺原氨酸；(A46) triiodothyronine;
(A47)茴三硫；(A47) Anethiol;
(A48)决奈达隆；(A48) Dronedarone;
(A49)他扎罗汀；(A49) Tazarotene;
(A50)6-巯基嘌呤；(A50) 6-mercaptopurine;
(A51)马拉硫磷；(A51) Malathion;
(A52)布托康唑；(A52) Butoconazole;
(A53)培西达替尼；(A53) Pecidinib;
(A54)溴化奥替洛宁；(A54) Oxilonine bromide;
(A55)硝羟喹啉；(A55) Nitroquinoline;
(A56)替莫泊芬；(A56) Temoporfin;
(A57)卡泊芬净；(A57) Caspofungin;
(A58)甲萘醌；(A58) menadione;
(A59)双醋瑞因；(A59) diacerein;
(A60)(R)-兰索拉唑；(A60)(R)-Lansoprazole;
(A61)恩贝酸；(A61) enbemic acid;
(A62)洛美他派；(A62) Lomitapide;
(A63)替拉那韦；(A63) Tipranavir;
(A64)索法酮；(A64) Sofalcone;
(A65)扎鲁司特；(A65) Zafirlukast;
(A66)兰索拉唑；(A66) lansoprazole;
(A67)雷洛昔芬；(A67) raloxifene;
(A68)特拉普韦；(A68) Trapuvir;
(A69)核黄素四丁酸酯；(A69) riboflavin tetrabutyrate;
(A70)左旋甲状腺素；(A70) Levothyroxine;
(A71)多库酯钠；(A71) docusate sodium;
(A72)决奈达隆；(A72) Dronedarone;
(A73)益康唑；(A73) Econazole;
(Z2)上述活性成分A1～A73的任一组合；(Z2) any combination of the above active ingredients A1 to A73;
其中，所述的活性成分或含所述活性成分的制剂被用于制备(a)抑制冠状病毒3CL蛋白酶的抑制剂；和/或(b)治疗和/或预防、缓解由冠状病毒(或单正链RNA病毒)感染引起的相关疾病的药物。Wherein, the active ingredient or the preparation containing the active ingredient is used to prepare (a) an inhibitor of coronavirus 3CL protease; and/or (b) a drug for treating and/or preventing and alleviating related diseases caused by coronavirus (or single positive-strand RNA virus) infection.
在另一优选例中，所述的药物还含有选自下组的一种或多种额外活性成分：In another preferred embodiment, the drug further contains one or more additional active ingredients selected from the following group:
(Y1)用于抑制病毒的中药制剂或其活性成分；(Y1) Traditional Chinese medicine preparations or their active ingredients for inhibiting viruses;
(Y2)RNA复制酶抑制剂(如Remdesivir(瑞德西韦或GS-5734))；(Y2) RNA replicase inhibitors (such as Remdesivir or GS-5734);
(Y3)洛匹那韦(Lopinavir)、利托那韦(Ritonavir)；(Y3) Lopinavir, Ritonavir;
(Y4)氯喹(Chloroquine,Sigma-C6628)、或其药学上可接受的盐；(Y4) Chloroquine (Sigma-C6628), or a pharmaceutically acceptable salt thereof;
(Y5)羟氯喹、或其药学上可接受的盐。(Y5) Hydroxychloroquine, or a pharmaceutically acceptable salt thereof.
在另一优选例中，所述的冠状病毒3CL蛋白酶选自下组：2019新型冠状病毒(SARS-CoV-2)3CL蛋白酶、SARS病毒的3CL蛋白酶、MERS病毒的3CL蛋白酶、或其组合。In another preferred embodiment, the coronavirus 3CL protease is selected from the following group: 2019 novel coronavirus (SARS-CoV-2) 3CL protease, SARS virus 3CL protease, MERS virus 3CL protease, or a combination thereof.
在另一优选例中，所述活性成分被用于制备(a)2019新型冠状病毒(SARS-CoV-2)3CL蛋白酶抑制剂；和/或(b)治疗和/或预防、缓解由2019新型冠状病毒(SARS-CoV-2)感染引起的相关疾病的药物。In another preferred embodiment, the active ingredient is used to prepare (a) a 2019 novel coronavirus (SARS-CoV-2) 3CL protease inhibitor; and/or (b) a drug for treating and/or preventing and alleviating related diseases caused by 2019 novel coronavirus (SARS-CoV-2) infection.
在另一优选例中，所述的冠状病毒选自下组：α属冠状病毒、β属冠状病毒、或其组合。In another preferred embodiment, the coronavirus is selected from the following group: alpha coronavirus, beta coronavirus, or a combination thereof.
在另一优选例中，所述的冠状病毒选自下组：HCoV-229E、HCoV-OC43、SARS-CoV、HCoV-NL63、HCoV-HKU1、MERS-CoV、SARS-CoV-2、或其组合。In another preferred embodiment, the coronavirus is selected from the group consisting of HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, SARS-CoV-2, or a combination thereof.
在另一优选例中，所述的冠状病毒选自下组：2019新型冠状病毒(SARS-CoV-2)、SARS病毒、MERS病毒、或其组合。In another preferred embodiment, the coronavirus is selected from the group consisting of 2019 novel coronavirus (SARS-CoV-2), SARS virus, MERS virus, or a combination thereof.
在另一优选例中，所述由2019新型冠状病毒感染引起的相关疾病选自下组：呼吸道感染、肺炎及其并发症、或其组合。In another preferred embodiment, the related diseases caused by 2019 novel coronavirus infection are selected from the following group: respiratory tract infection, pneumonia and its complications, or a combination thereof.
在另一优选例中，所述活性成分选自表1中的活性化合物。In another preferred embodiment, the active ingredient is selected from the active compounds in Table 1.
在另一优选例中，所述的活性成分选自下组：选自A1～A18中任一活性成分(老药)、或其药学上可接受的盐或其提取物。In another preferred embodiment, the active ingredient is selected from the following group: any active ingredient selected from A1 to A18 (old medicine), or a pharmaceutically acceptable salt thereof or an extract thereof.
在另一优选例中，所述的活性成分选自下组：金诺芬、雷贝拉唑钠、肝素钠、双硫仑、艾普拉唑、博赛泼维、泰妥拉唑、或其药学上可接受的盐。In another preferred embodiment, the active ingredient is selected from the following group: auranofin, rabeprazole sodium, heparin sodium, disulfiram, ilaprazole, boceprevir, tenatoprazole, or a pharmaceutically acceptable salt thereof.
在另一优选例中，所述的活性成分(或活性化合物)是人工合成的、和/或从植物中提取的。In another preferred embodiment, the active ingredient (or active compound) is artificially synthesized and/or extracted from plants.
在另一优选例中，所述的植物或药材包括根、茎、叶、花、果、或其组合。In another preferred embodiment, the plant or medicinal material includes roots, stems, leaves, flowers, fruits, or a combination thereof.
在另一优选例中，所述的药物还包括选自下组的额外组分：抗逆传录病毒的药物或增强免疫力的药物。In another preferred embodiment, the drug further comprises an additional component selected from the following group: an antiretroviral drug or an immunity enhancing drug.
在另一优选例中，所述的组合物或药物包括：口服制剂和非口服制剂。In another preferred embodiment, the composition or medicine includes: an oral preparation and a parenteral preparation.
在另一优选例中，所述的制剂包括：粉剂、颗粒剂、胶囊剂、注射剂、酊剂、口服液、片剂、含片、或滴丸。In another preferred embodiment, the preparation includes: powder, granules, capsules, injections, tinctures, oral liquids, tablets, lozenges, or pills.
在本发明的第二方面，提供了一种药物组合物，所述的药物组合物含有：In a second aspect of the present invention, a pharmaceutical composition is provided, comprising:
(a)第一活性成分，所述的第一活性成分选自下组：A1～A73中任一活性成分(老药)、或其药学上可接受的盐或其提取物；或其组合；(a) a first active ingredient, wherein the first active ingredient is selected from the following group: any active ingredient A1 to A73 (old medicine), or a pharmaceutically acceptable salt or extract thereof; or a combination thereof;
以及(b)药学上可接受的载体。and (b) a pharmaceutically acceptable carrier.
在另一优选例中，所述的药物组合物为用于抑制冠状病毒3CL蛋白酶的药物组合物。In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition for inhibiting coronavirus 3CL protease.
在另一优选例中，所述的药物组合物中，除了选自A1～A73中任一活性成分(老药)、或其药学上可接受的盐或其提取物之外，不含有其他活性成分(如抗病毒的活性成分)。In another preferred embodiment, the pharmaceutical composition does not contain other active ingredients (such as antiviral active ingredients) except any active ingredient (old drug) selected from A1 to A73, or a pharmaceutically acceptable salt or an extract thereof.
在另一优选例中，所述的药物组合物含有：In another preferred embodiment, the pharmaceutical composition contains:
(a1)第一活性成分，所述的第一活性成分选自A1～A73中任一活性成分(老药)、或其药学上可接受的盐或其提取物；或其组合；和(a1) a first active ingredient, wherein the first active ingredient is selected from any active ingredient A1 to A73 (old medicine), or a pharmaceutically acceptable salt or extract thereof; or a combination thereof; and
(a2)第二活性成分，所述的第二活性成分选自下组：RNA复制酶抑制剂(如Remdesivir(瑞德西韦或GS-5734))；洛匹那韦(Lopinavir)、利托那韦(Ritonavir)；氯喹(Chloroquine,Sigma-C6628)、羟氯喹、或其组合；(a2) a second active ingredient, wherein the second active ingredient is selected from the following group: RNA replicase inhibitors (such as Remdesivir or GS-5734); Lopinavir, Ritonavir; Chloroquine (Sigma-C6628), hydroxychloroquine, or a combination thereof;
以及(b)药学上可接受的载体。and (b) a pharmaceutically acceptable carrier.
在另一优选例中，所述的药物的剂型为口服给药或非口服给药剂型。In another preferred embodiment, the dosage form of the drug is an oral or parenteral dosage form.
在另一优选例中，所述的口服给药剂型是片剂、散剂、颗粒剂或胶囊剂，或乳剂或糖浆剂。In another preferred embodiment, the oral dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.
在另一优选例中，所述的非口服给药剂型是注射剂或针剂。In another preferred embodiment, the non-oral dosage form is an injection or injection.
在本发明的第三方面，提供了本发明第二方面中所述的药物组合物的用途，它被用于制备(a)抑制冠状病毒3CL蛋白酶的抑制剂；和/或(b)治疗和/或预防、缓解由冠状病毒感染引起的相关疾病的药物。In the third aspect of the present invention, the use of the pharmaceutical composition described in the second aspect of the present invention is provided, which is used to prepare (a) an inhibitor of coronavirus 3CL protease; and/or (b) a drug for treating and/or preventing and alleviating related diseases caused by coronavirus infection.
在另一优选例中，所述药物组合物用于制备治疗和/或预防、缓解由2019新型冠状病毒(SARS-CoV-2)感染引起的相关疾病的药物。In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for treating and/or preventing and alleviating related diseases caused by infection with the 2019 novel coronavirus (SARS-CoV-2).
在另一优选例中，所述由2019新型冠状病毒感染引起的相关疾病选自下组：呼吸道感染、肺炎及其并发症、或其组合。In another preferred embodiment, the related diseases caused by 2019 novel coronavirus infection are selected from the following group: respiratory tract infection, pneumonia and its complications, or a combination thereof.
在另一优选例中，所述的冠状病毒选自下组：HCoV-229E、HCoV-OC43、SARS-CoV、HCoV-NL63、HCoV-HKU1、MERS-CoV、SARS-CoV-2、或其组合。In another preferred embodiment, the coronavirus is selected from the group consisting of HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, SARS-CoV-2, or a combination thereof.
在本发明的第四方面，提供了一种抑制冠状病毒3CL蛋白酶的方法，包括步骤：将第一活性成分或含所述第一活性成分的制剂与冠状病毒的3CL蛋白酶接触，从而抑制所述3CL蛋白酶的活性；In a fourth aspect of the present invention, a method for inhibiting coronavirus 3CL protease is provided, comprising the steps of: contacting a first active ingredient or a preparation containing the first active ingredient with a coronavirus 3CL protease, thereby inhibiting the activity of the 3CL protease;
其中，所述的第一活性成分选自下组：Wherein, the first active ingredient is selected from the following group:
(Z1)选自A1～A73中任一活性成分(老药)、或其药学上可接受的盐或其提取物；(Z1) any active ingredient selected from A1 to A73 (old drug), or a pharmaceutically acceptable salt or extract thereof;
(Z2)上述活性成分A1～A73的任一组合。(Z2) Any combination of the above active ingredients A1 to A73.
在另一优选例中，所述的抑制方法是体外方法并且是非治疗性的和非诊断性的方法。In another preferred embodiment, the inhibition method is an in vitro method and is a non-therapeutic and non-diagnostic method.
在另一优选例中，所述的抑制方法是体内方法并且是治疗性的方法。In another preferred embodiment, the inhibition method is an in vivo method and a therapeutic method.
在另一优选例中，所述的方法是非治疗性和非诊断性的。In another preferred embodiment, the method is non-therapeutic and non-diagnostic.
在另一优选例中，所述的方法是体外的。In another preferred embodiment, the method is in vitro.
在另一优选例中，所述的3CL蛋白酶是重组的或冠状病毒表达的3CL蛋白酶。In another preferred embodiment, the 3CL protease is a recombinant or coronavirus-expressed 3CL protease.
在另一优选例中，所述的SARS-CoV-2的3CL蛋白酶是重组的或SARS-CoV-2表达的3CL蛋白酶。In another preferred embodiment, the SARS-CoV-2 3CL protease is a recombinant or SARS-CoV-2 expressed 3CL protease.
在另一优选例中，所述的第一活性成分选自下组：A1～A18中任一活性成分、或其药学上可接受的盐或其提取物；或其组合。In another preferred embodiment, the first active ingredient is selected from the following group: any active ingredient of A1 to A18, or a pharmaceutically acceptable salt or extract thereof; or a combination thereof.
在另一优选例中，所述的第一活性成分选自下组：A1～A10中任一活性成分、或其药学上可接受的盐或其提取物；或其组合。In another preferred embodiment, the first active ingredient is selected from the following group: any active ingredient of A1 to A10, or a pharmaceutically acceptable salt or extract thereof; or a combination thereof.
在另一优选例中，所述的第一活性成分选自下组：金诺芬、雷贝拉唑钠、肝素钠、双硫仑、艾普拉唑、博赛泼维、泰妥拉唑、或其药学上可接受的盐；或其组合。In another preferred embodiment, the first active ingredient is selected from the following group: auranofin, rabeprazole sodium, heparin sodium, disulfiram, ilaprazole, boceprevir, tenatoprazole, or a pharmaceutically acceptable salt thereof; or a combination thereof.
在本发明的第五方面，提供了一种治疗、预防、和/或缓解由冠状病毒感染引起的相关疾病的方法，包括步骤：给需要的对象施用安全有效量的第一活性成分或含第一活性成分的制剂，其中所述的第一活性成分选自下组：In a fifth aspect of the present invention, a method for treating, preventing, and/or alleviating related diseases caused by coronavirus infection is provided, comprising the steps of administering a safe and effective amount of a first active ingredient or a preparation containing the first active ingredient to a subject in need thereof, wherein the first active ingredient is selected from the group consisting of:
(Z1)选自A1～A73中任一活性成分(老药)、或其药学上可接受的盐或其提取物；(Z1) any active ingredient selected from A1 to A73 (old drug), or a pharmaceutically acceptable salt or extract thereof;
(Z2)上述活性成分A1～A73的任一组合。(Z2) Any combination of the above active ingredients A1 to A73.
在另一优选例中，所述的制剂为非中药制剂。In another preferred embodiment, the preparation is a non-traditional Chinese medicine preparation.
在另一优选例中，所述方法还包括：给需要的对象施用安全有效量的第二活性成分和任选的第三活性成分，其中In another preferred embodiment, the method further comprises: administering a safe and effective amount of a second active ingredient and an optional third active ingredient to a subject in need thereof, wherein
所述的第二活性成分选自下组：RNA复制酶抑制剂(如Remdesivir(瑞德西韦或GS-5734))；洛匹那韦(Lopinavir)、利托那韦(Ritonavir)；氯喹(Chloroquine,Sigma-C6628)、羟氯喹、或其组合；The second active ingredient is selected from the following group: RNA replicase inhibitors (such as Remdesivir or GS-5734); Lopinavir, Ritonavir; Chloroquine (Sigma-C6628), hydroxychloroquine, or a combination thereof;
在另一优选例中，所述的对象为哺乳动物，较佳地为灵长目哺乳动物，更佳地为人。In another preferred embodiment, the subject is a mammal, preferably a primate, and more preferably a human.
在另一优选例中，所述的第一活性成分选自下组：A1～A18中任一活性成分、或其药学上可接受的盐或其提取物；或其组合。In another preferred embodiment, the first active ingredient is selected from the following group: any active ingredient of A1 to A18, or a pharmaceutically acceptable salt or extract thereof; or a combination thereof.
在另一优选例中，所述的第一活性成分选自下组：A1～A10中任一活性成分、或其药学上可接受的盐或其提取物；或其组合。In another preferred embodiment, the first active ingredient is selected from the following group: any active ingredient of A1 to A10, or a pharmaceutically acceptable salt or extract thereof; or a combination thereof.
在另一优选例中，所述的第一活性成分选自下组：金诺芬、雷贝拉唑钠、肝素钠、双硫仑、艾普拉唑、博赛泼维、泰妥拉唑、或其药学上可接受的盐；或其组合。In another preferred embodiment, the first active ingredient is selected from the following group: auranofin, rabeprazole sodium, heparin sodium, disulfiram, ilaprazole, boceprevir, tenatoprazole, or a pharmaceutically acceptable salt thereof; or a combination thereof.
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1显示了金诺芬浓度依赖的抑制2019新型冠状病毒(SARS-CoV-2)3CL蛋白酶(SARS-CoV-2-3CLpro)酶活的曲线。Figure 1 shows the curve of the inhibition of the activity of 2019 novel coronavirus (SARS-CoV-2) 3CL protease (SARS-CoV-2-3CLpro) by auranofin in a concentration-dependent manner.
图2显示了雷贝拉唑钠浓度依赖的抑制SARS-CoV-2-3CLpro酶活的曲线。Figure 2 shows the curve of the concentration-dependent inhibition of SARS-CoV-2-3CLpro enzyme activity by rabeprazole sodium.
图3显示了肝素钠浓度依赖的抑制SARS-CoV-2-3CLpro酶活的曲线。Figure 3 shows the curve of heparin sodium concentration-dependent inhibition of SARS-CoV-2-3CLpro enzyme activity.
图4显示了双硫仑浓度依赖的抑制SARS-CoV-2-3CLpro酶活的曲线。Figure 4 shows the curve of disulfiram concentration-dependent inhibition of SARS-CoV-2-3CLpro enzyme activity.
图5显示了艾普拉唑浓度依赖的抑制SARS-CoV-2-3CLpro酶活的曲线。FIG5 shows the curve of ilaprazole concentration-dependent inhibition of SARS-CoV-2-3 CLpro enzyme activity.
图6显示了博赛泼维浓度依赖的抑制SARS-CoV-2-3CLpro酶活的曲线。FIG6 shows the curve of the concentration-dependent inhibition of SARS-CoV-2-3CLpro enzyme activity by boceprevir.
图7显示了泰妥拉唑浓度依赖的抑制SARS-CoV-2-3CLpro酶活的曲线。Figure 7 shows the curve of the inhibition of SARS-CoV-2-3CLpro enzyme activity by tenotoprazole concentration-dependently.
具体实施方式DETAILED DESCRIPTION
本发明人经过广泛而深入的研究，通过筛选已批准上市的2000个药物分子，首次意外地开发了有效抑制2019新型冠状病毒(SARS-CoV-2)等冠状病毒的3CL蛋白酶的活性的金诺芬等多个老药。实验表明，本发明的活性分子(如金诺芬等多个老药或其药学上可接受的盐)可高效地抑制2019新型冠状病毒(SARS-CoV-2)等冠状病毒的3CL蛋白酶的活性，从而抑制SARS-CoV-2冠状病毒的复制和活力。在此基础上完成了本发明。After extensive and in-depth research, the inventors unexpectedly developed a number of old drugs such as Auranofin that effectively inhibit the activity of 3CL proteases of coronaviruses such as the 2019 novel coronavirus (SARS-CoV-2) by screening 2,000 drug molecules that have been approved for marketing. Experiments have shown that the active molecules of the present invention (such as auranofin and other old drugs or their pharmaceutically acceptable salts) can effectively inhibit the activity of 3CL proteases of coronaviruses such as the 2019 novel coronavirus (SARS-CoV-2), thereby inhibiting the replication and activity of SARS-CoV-2 coronavirus. The present invention was completed on this basis.
具体地，本发明揭示了金诺芬等多个老药及其组合物在抗单正链RNA病毒的用途，尤其是在抗COVID-19治疗中的用途。金诺芬等多个老药及其组合物对冠状病毒复制必不可少且高度保守的3CL水解酶具有优良的抑制作用，具有良好的临床应用前景。Specifically, the present invention discloses the use of auranofin and other old drugs and their compositions in the treatment of single positive-strand RNA viruses, especially in the treatment of COVID-19. Auranofin and other old drugs and their compositions have excellent inhibitory effects on 3CL hydrolases, which are essential for coronavirus replication and highly conserved, and have good clinical application prospects.
术语the term
如本文所用，“本发明的活性化合物”、“本发明的抑制3CL蛋白酶的活性化合物”可互换使用，指具有优异的3CL蛋白酶抑制活性的化合物，尤其是金诺芬等已知药物活性成分(老药)或其组合。As used herein, "active compounds of the present invention" and "active compounds for inhibiting 3CL proteases of the present invention" are used interchangeably and refer to compounds having excellent 3CL protease inhibitory activity, especially known active pharmaceutical ingredients (old drugs) such as auranofin or combinations thereof.
如本文所用，“本发明的药材”指含有本发明活性化合物的中药材。As used herein, "the medicinal material of the present invention" refers to the Chinese medicinal material containing the active compound of the present invention.
如本文所用，“本发明的药材提取物”或“本发明的提取物”指用中药材或相应植物提取所获得的且含有本发明的一种或多种活性化合物的提取物。As used herein, "the medicinal material extract of the present invention" or "the extract of the present invention" refers to an extract obtained by extracting Chinese medicinal materials or corresponding plants and containing one or more active compounds of the present invention.
如本文所用，“本发明的制剂”指含有本发明活性化合物的制剂，包括中药制剂和非中药制剂。As used herein, "the preparation of the present invention" refers to a preparation containing the active compound of the present invention, including traditional Chinese medicine preparations and non-traditional Chinese medicine preparations.
如本文所用，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所述的元件或组成部分，而并未排除其它元件或其它组成部分。As used herein, the term “comprise” or variations thereof such as “include” or “comprising”, etc., will be understood as including the stated elements or components but not excluding other elements or components.
冠状病毒和3CL蛋白酶Coronavirus and 3CL protease
冠状病毒(Coronavirus，CoV)属于套式病毒目(Nidovirales)冠状病毒科(Coronaviridae)，是一种有包膜的正链RNA病毒，其亚科包含α、β、δ及γ四属。Coronavirus (CoV) belongs to the Coronaviridae family of the order Nidovirales. It is an enveloped positive-strand RNA virus, and its subfamily includes four genera: α, β, δ, and γ.
目前已知的感染人的冠状病毒中，HCoV-229E和HCoV-NL63属于α属冠状病毒，HCoV-OC43、SARS-CoV、HCoV-HKU1、MERS-CoV和SARS-CoV-2均为β属冠状病毒。Among the currently known coronaviruses that infect humans, HCoV-229E and HCoV-NL63 belong to the alpha coronavirus family, while HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and SARS-CoV-2 are all beta coronaviruses.
高致病性冠状病毒“非典”SARS-CoV和“中东呼吸综合征”MERS-CoV均属于β属冠状病毒。新型冠状病毒(SARS-CoV-2)与SARS-CoV有约80％相似性、与MERS-CoV有40％的相似性，也属于β属冠状病毒。Highly pathogenic coronaviruses SARS-CoV and MERS-CoV are both beta coronaviruses. The new coronavirus (SARS-CoV-2) is about 80% similar to SARS-CoV and 40% similar to MERS-CoV, and is also a beta coronavirus.
该类病毒的基因组是一条单股正链RNA，是基因组最大的RNA病毒之一，编码包括复制酶、刺突蛋白、囊膜蛋白、包膜蛋白和核壳蛋白等。在病毒复制的初始阶段，基因组被翻译成两条长达几千个氨基酸的肽链即前体多聚蛋白(Polyprotein)，随后前体蛋白被蛋白酶切割生成非结构蛋白(如RNA聚合酶和解旋酶)和结构蛋白(如刺突蛋白)及辅助蛋白。The genome of this type of virus is a single-stranded positive-strand RNA, which is one of the largest RNA viruses. It encodes replicase, spike protein, envelope protein, envelope protein and nucleocapsid protein, etc. In the initial stage of virus replication, the genome is translated into two peptide chains of several thousand amino acids, namely precursor polyproteins. Subsequently, the precursor proteins are cleaved by proteases to generate non-structural proteins (such as RNA polymerase and helicase) and structural proteins (such as spike protein) and auxiliary proteins.
3CL蛋白酶(3Chymotrypsin-like protease,3CLpro)是冠状病毒中负责切割前体蛋白的主蛋白酶(所以又称为Mpro)，对病毒的复制不可或缺。3CL protease (3Chymotrypsin-like protease, 3CLpro) is the main protease responsible for cleaving precursor proteins in coronaviruses (so it is also called Mpro ), and is indispensable for viral replication.
3CLpro属于半胱氨酸水解酶，在各类冠状病毒中高度保守，与小RNA病毒中的3C蛋白酶也比较相似，而人体中却没有与其相似的蛋白酶，因而是研发广谱抗单正链RNA病毒药物的理想靶点。3CLpro is a cysteine hydrolase that is highly conserved among various coronaviruses and is similar to the 3C protease in small RNA viruses. However, there is no similar protease in the human body, making it an ideal target for the development of broad-spectrum anti-single positive-strand RNA virus drugs.
本发明的活性化合物和活性成分Active compounds and active ingredients of the invention
在本发明中，提供了一种可有效抑制2019新型冠状病毒(SARS-CoV-2)等冠状病毒复制的活性成分。该活性成分选自下组：In the present invention, an active ingredient is provided that can effectively inhibit the replication of coronaviruses such as the 2019 novel coronavirus (SARS-CoV-2). The active ingredient is selected from the following group:
(Z1)选自A1～A73中任一活性成分、或其药学上可接受的盐或其提取物；(Z1) any active ingredient selected from A1 to A73, or a pharmaceutically acceptable salt or extract thereof;
(Z2)上述活性成分A1～A73的任一组合(包括上述活性成分Z1中两种或多种成分的任意组合)。(Z2) Any combination of the above active ingredients A1 to A73 (including any combination of two or more of the above active ingredients Z1).
试验表明，本发明的活性化合物可有效地抑制2019新型冠状病毒(SARS-CoV-2)的3CL蛋白酶，从而抑制2019新型冠状病毒(SARS-CoV-2)的复制，进而预防、治疗和/或缓解SARS-CoV-2相关疾病。Experiments have shown that the active compounds of the present invention can effectively inhibit the 3CL protease of the 2019 novel coronavirus (SARS-CoV-2), thereby inhibiting the replication of the 2019 novel coronavirus (SARS-CoV-2), and further preventing, treating and/or alleviating SARS-CoV-2 related diseases.
如本文所用，“本发明的活性化合物”、“本发明的抑制3CL蛋白酶的活性化合物”可互换使用，指具有优异的3CL蛋白酶抑制活性的化合物，尤其是以金诺芬为代表的A1～A73中任一活性成分、或其组合。As used herein, "active compound of the present invention" and "active compound for inhibiting 3CL protease of the present invention" are used interchangeably and refer to compounds with excellent 3CL protease inhibitory activity, especially any active ingredient A1 to A73 represented by auranofin, or a combination thereof.
应理解，本发明的活性成分包括本发明的抑制3CL蛋白酶的活性化合物、或其药学上可接受的盐、对映异构体、非对映异构体或外消旋体、或其前药。应理解，本发明的活性成分还包括本发明的活性化合物的晶型、无定形化合物、、氘代化合物、溶剂化物、水合物等形式。It should be understood that the active ingredients of the present invention include the active compounds of the present invention that inhibit 3CL proteases, or pharmaceutically acceptable salts, enantiomers, diastereomers or racemates thereof, or prodrugs thereof. It should be understood that the active ingredients of the present invention also include crystalline forms, amorphous compounds, deuterated compounds, solvates, hydrates, and the like of the active compounds of the present invention.
所述“药学上可接受的盐”为本发明的活性化合物与无机酸或有机酸反应形成常规的无毒盐。例如，常规的无毒盐可通过本发明的活性化合物与无机酸或有机酸反应制得，所述无机酸包括盐酸、氢溴酸、硫酸、硝酸、胺基磺酸和磷酸等，所述有机酸包括柠檬酸、酒石酸、乳酸、丙酮酸、乙酸、苯磺酸、对甲苯磺酸、甲磺酸、萘磺酸、乙磺酸、萘二磺酸、马来酸、苹果酸、丙二酸、富马酸、琥珀酸、丙酸、草酸、三氟乙酸、硬酯酸、扑酸、羟基马来酸、苯乙酸、苯甲酸、水杨酸、谷氨酸、抗坏血酸、对胺基苯磺酸、2-乙酰氧基苯甲酸和羟乙磺酸等；或者本发明的活性化合物与丙酸、草酸、丙二酸、琥珀酸、富马酸、马来酸、乳酸、苹果酸、酒石酸、柠檬酸、天冬氨酸或谷氨酸形成酯后再与无机碱形成的钠盐、钾盐、钙盐、铝盐或铵盐；或者本发明的活性化合物与有机碱形成的甲胺盐、乙胺盐或乙醇胺盐；或者本发明的活性化合物与赖氨酸、精氨酸、鸟氨酸形成酯后再与盐酸、氢溴酸、氢氟酸、硫酸、硝酸或磷酸形成的对应的无机酸盐或与甲酸、乙酸、苦味酸、甲磺酸或乙磺酸形成的对应的有机酸盐。The "pharmaceutically acceptable salt" is a conventional non-toxic salt formed by the reaction of the active compound of the present invention with an inorganic acid or an organic acid. For example, conventional non-toxic salts can be prepared by reacting the active compound of the present invention with an inorganic acid or an organic acid, wherein the inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid and phosphoric acid, and the organic acid includes citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid, and the like; Or the active compound of the present invention forms an ester with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid or glutamic acid, and then forms a sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt with an inorganic base; or the active compound of the present invention forms a methylamine salt, ethylamine salt or ethanolamine salt with an organic base; or the active compound of the present invention forms an ester with lysine, arginine, or ornithine, and then forms a corresponding inorganic acid salt with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid or phosphoric acid, or a corresponding organic acid salt with formic acid, acetic acid, picric acid, methanesulfonic acid or ethanesulfonic acid.
此外，本发明的活性成分还特别适合与其他抗病毒的药物联用。代表性的其他的抗病毒药物包括(但并不限于)：逆转录酶抑制剂、蛋白酶抑制剂、辅助受体拮抗剂、逆转录病毒整合酶抑制剂、病毒吸附抑制剂、特异性病毒转录抑制剂、抗体、或其组合。In addition, the active ingredients of the present invention are particularly suitable for use in combination with other antiviral drugs. Representative other antiviral drugs include (but are not limited to): reverse transcriptase inhibitors, protease inhibitors, coreceptor antagonists, retroviral integrase inhibitors, viral adsorption inhibitors, specific viral transcription inhibitors, antibodies, or combinations thereof.
本发明的活性成分可抑制SARS-CoV-2等新型冠状病毒的感染活性。因此，当在治疗上施用或给予本发明的活性成分时，可抑制3CL蛋白酶活性，从而抑制2019新型冠状病毒(SARS-CoV-2)的感染，进而达到抗病毒作用。The active ingredients of the present invention can inhibit the infection activity of new coronaviruses such as SARS-CoV-2. Therefore, when the active ingredients of the present invention are applied or administered therapeutically, the activity of 3CL protease can be inhibited, thereby inhibiting the infection of the 2019 new coronavirus (SARS-CoV-2), thereby achieving an antiviral effect.
药物组合物和应用Pharmaceutical compositions and applications
本发明还提供了以本发明的抑制3CL蛋白酶的活性化合物、或其药学上可接受的盐、或其前药、或其提取物、或其药材中的一种或多种的混合物为有效成分在制备治疗和/或预防、缓解由2019新型冠状病毒感染引起的呼吸道感染、肺炎等相关疾病的药物中的用途。The present invention also provides the use of the active compound for inhibiting 3CL protease of the present invention, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or an extract thereof, or a mixture of one or more of its medicinal materials as an effective ingredient in the preparation of a drug for treating and/or preventing or alleviating respiratory tract infections, pneumonia and other related diseases caused by 2019 novel coronavirus infection.
本发明所提供的药物组合物优选含有重量比为0.001-99wt％的活性成份，优选的比例是本发明的活性化合物作为活性成分占总重量的0.1wt％～90wt％或1wt％～50wt％，其余部分为药学可接受的载体、稀释液或溶液或盐溶液。The pharmaceutical composition provided by the present invention preferably contains an active ingredient in a weight ratio of 0.001-99wt%, preferably a ratio in which the active compound of the present invention as an active ingredient accounts for 0.1wt% to 90wt% or 1wt% to 50wt% of the total weight, and the remainder is a pharmaceutically acceptable carrier, diluent or solution or saline solution.
需要的时候，在本发明药物中还可以加入一种或多种药学上可接受的载体。所述载体包括药学领域常规的稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。When necessary, one or more pharmaceutically acceptable carriers may be added to the drug of the present invention, including conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.
本发明所提供的化合物和药物组合物可以是多种形式，如片剂、胶囊、粉剂、糖浆、溶液状、悬浮液和气雾剂等，并可以存在于适宜的固体或液体的载体或稀释液中和适宜的用于注射或滴注的消毒器具中。The compounds and pharmaceutical compositions provided by the present invention may be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, and may be present in suitable solid or liquid carriers or diluents and in suitable sterile devices for injection or infusion.
本发明的药物组合物的各种剂型可按照药学领域的常规制备方法制备。其制剂配方的单位计量中通常包含0.05-400mg本发明的活性化合物，优选地，制剂配方的单位计量中包含1mg-500mg本发明的活性化合物。The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dosage of the preparation formula generally contains 0.05-400 mg of the active compound of the present invention, preferably, the unit dosage of the preparation formula contains 1 mg-500 mg of the active compound of the present invention.
本发明的化合物和药物组合物可对哺乳动物临床使用，包括人和动物，可以通过口、鼻、皮肤、肺或者胃肠道等的给药途径。最优选为口服。最优选日剂量为0.01-400mg/kg体重，一次性服用，或0.01-200mg/kg体重分次服用。不管用何种服用方法，个人的最佳剂量应依据具体的治疗而定。通常情况下是从小剂量开始，逐渐增加剂量一直到找到最适合的剂量。The compounds and pharmaceutical compositions of the present invention can be used clinically in mammals, including humans and animals, and can be administered by oral, nasal, skin, lung or gastrointestinal tract administration. Oral administration is most preferred. The most preferred daily dose is 0.01-400 mg/kg body weight, taken at one time, or 0.01-200 mg/kg body weight taken in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment. Usually, a small dose is started and the dose is gradually increased until the most suitable dose is found.
本发明的药物或抑制剂可通过各种不同方式施用，例如可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、静脉、粘膜组织；或是被其他物质混合或包裹导入机体。The drugs or inhibitors of the present invention can be administered in various ways, for example, by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods, such as introduction into the body into muscle, intradermal, subcutaneous, intravenous, mucosal tissue; or mixed or encapsulated in other substances and introduced into the body.
本发明的主要优点包括：The main advantages of the present invention include:
(a)本发明活性化合物可高效地抑制SARS-CoV-2 3CL蛋白酶，部分活性化合物的IC50值达低于5μM或更低。(a) The active compounds of the present invention can effectively inhibit SARS-CoV-2 3CL protease, and theIC50 values of some active compounds are less than 5 μM or lower.
(b)由于各种冠状病毒中3CLpro及其底物结合口袋的高度保守性，本发明活性化合物有望抑制其他冠状病毒3CLpro而发挥广谱的抗病毒活性。(b) Due to the high conservation of 3CLpro and its substrate binding pocket in various coronaviruses, the active compounds of the present invention are expected to inhibit other coronavirus 3CLpro and exert broad-spectrum antiviral activity.
(c)本发明活性化合物都是老药，毒副作用低，成药性好。(c) The active compounds of the present invention are all old drugs with low toxicity and side effects and good drugability.
(d)本发明的技术平台能够快速有效的发现冠状病毒3CL水解酶的抑制剂，并应用该方法发现了金诺芬等多个老药及其组合物对SARS-CoV-2病毒3CL水解酶的抑制作用。(d) The technical platform of the present invention can quickly and effectively discover inhibitors of coronavirus 3CL hydrolase, and the method was used to discover the inhibitory effects of several old drugs such as auranofin and their compositions on SARS-CoV-2 virus 3CL hydrolase.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.
样品的分析数据由以下仪器测定：核磁共振由GEMINI-300型、Bruker AMX-400型和INVOA-600型核磁共振仪测定，TMS(四甲基硅烷)为内标，化学位移单位为ppm，耦合常数单位为Hz；质谱由Finnigan MAT-711型，MAT-95和LCQ-DECA型质谱仪以及IonSpec4.7Tesla质谱仪测定。The analytical data of the samples were measured by the following instruments: nuclear magnetic resonance was measured by GEMINI-300, Bruker AMX-400 and INVOA-600 nuclear magnetic resonance instruments, TMS (tetramethylsilane) was used as the internal standard, the chemical shift unit was ppm, and the coupling constant unit was Hz; mass spectrometry was measured by Finnigan MAT-711, MAT-95 and LCQ-DECA mass spectrometers and IonSpec 4.7 Tesla mass spectrometer.
柱层析用硅胶200-300目(青岛海洋化工厂生产)；TLC硅胶板为烟台化工厂生产的HSGF-254型薄层层析预制板；石油醚沸程为60-90℃；采用紫外灯，碘缸显色。除另有说明外，以下实施例中所用常规试剂、药品均购自国药集团。实验中所用试剂及溶剂均按反应具体情况处理。Silica gel 200-300 mesh (produced by Qingdao Ocean Chemical Plant) was used for column chromatography; TLC silica gel plate was HSGF-254 thin layer chromatography prefabricated plate produced by Yantai Chemical Plant; petroleum ether boiling range was 60-90°C; ultraviolet lamp and iodine cylinder were used for color development. Unless otherwise specified, conventional reagents and drugs used in the following examples were purchased from Sinopharm Group. The reagents and solvents used in the experiment were handled according to the specific conditions of the reaction.
实施例1：SARS-CoV-2-3CLpro抑制剂发现方法的建立Example 1: Establishment of a method for discovering SARS-CoV-2-3CLpro inhibitors
利用荧光共振能量转移方法评价测定单个化合物及混合物对SARS-CoV-23CLpro酶活的抑制活性。整个酶促反应体系的体积为120μL，蛋白酶的终浓度为30nM，底物终浓度为20μM。反应体系的缓冲液包括50mM Tris pH7.3、1mM EDTA。在96孔板中加入SARS-CoV-23CLpro蛋白酶和不同浓度的化合物或混合物等样品，30℃孵育10min，加入底物并迅速放入酶标仪中读数。激发光和发射光分别为340nm和405nm。测试时间为10min，每隔30s读一次荧光值。最终结果取前2min的读值拟合出反应速率，并与对照组(DMSO)比较，计算抑制率。利用软件GraphPad Prism 8拟合得到IC50值。The fluorescence resonance energy transfer method was used to evaluate the inhibitory activity of individual compounds and mixtures on SARS-CoV-23CLpro enzyme activity. The volume of the entire enzymatic reaction system was 120 μL, the final concentration of the protease was 30 nM, and the final concentration of the substrate was 20 μM. The buffer of the reaction system included 50 mM Tris pH 7.3 and 1 mM EDTA. SARS-CoV-23CLpro protease and samples such as compounds or mixtures of different concentrations were added to a 96-well plate, incubated at 30 ° C for 10 min, and the substrate was added and quickly placed in a microplate reader for reading. The excitation light and emission light were 340 nm and 405 nm, respectively. The test time was 10 min, and the fluorescence value was read every 30 s. The final result was to fit the reaction rate with the reading of the first 2 min, and compared with the control group (DMSO) to calculate the inhibition rate. The IC50 value was obtained by fitting using the software GraphPad Prism 8.
该方法简便、灵敏，可高通量筛选SARS-CoV-2-3CLpro的抑制剂(包括单体和混合物等各种实体样品)。此外，由于各种冠状病毒中3CLpro及其底物结合口袋的高度保守性，该方法也适用于其他冠状病毒3CLpro如SARS-nCoV-3CLpro抑制剂的发现。This method is simple, sensitive, and can be used for high-throughput screening of SARS-CoV-2-3CLpro inhibitors (including various solid samples such as monomers and mixtures). In addition, due to the high conservation of 3CLpro and its substrate binding pocket in various coronaviruses, this method is also applicable to the discovery of inhibitors of other coronavirus 3CLpro such as SARS-nCoV-3CLpro.
实施例2：金诺芬等多个老药对SARS-CoV-2-3CLpro的抑制作用测定Example 2: Determination of the inhibitory effect of several old drugs such as Auranofin on SARS-CoV-2-3CLpro
本实施例中所测试2000个老药通过购买获得，确证其结构和纯度(＞90％)。化合物先溶于DMSO配成母液，采用酶活测试缓冲液稀释DMSO母液配置各浓度化合物样品。The 2000 old drugs tested in this example were purchased, and their structures and purity (>90%) were confirmed. The compound was first dissolved in DMSO to prepare a mother solution, and the DMSO mother solution was diluted with enzyme activity test buffer to prepare compound samples of various concentrations.
采用实施例1中建立的SARS-CoV-2-3CLpro抑制剂发现方法，对2000个老药库中的化合物(金诺芬等多个老药)对SARS-CoV-2-3CLpro的抑制作用进行测定，化合物不同浓度对SARS-CoV-2-3CLpro的抑制率及IC50如表1所示。The SARS-CoV-2-3CLpro inhibitor discovery method established in Example 1 was used to determine the inhibitory effects of compounds in 2000 old drug libraries (multiple old drugs such as Auranofin) on SARS-CoV-2-3CLpro. The inhibition rates andIC50 of different concentrations of the compounds on SARS-CoV-2-3CLpro are shown in Table 1.
实验结果如表1所示。The experimental results are shown in Table 1.
表1.金诺芬等多个老药对2019新型冠状病毒3CL蛋白酶抑制活性Table 1. Inhibitory activity of auranofin and other old drugs against 2019 novel coronavirus 3CL protease
结果表明，金诺芬等多个老药(A1～A73)对SARS-CoV-2-3CLpro具有极其显著的抑制作用(表1)。金诺芬等多个老药均为新发现的老药来源的SARS-CoV-2-3CLpro抑制剂，IC50达到个位数微摩尔级别，最优化合物金诺芬、雷贝拉唑钠、肝素钠、双硫仑、艾普拉唑、博赛泼维和泰妥拉唑等的IC50分别为1.1μM、1.5μM、1.5μM、1.6μM、2.6μM、4.8μM和6.5μM(图1-7)。The results showed that several old drugs such as Auranofin (A1-A73) have extremely significant inhibitory effects on SARS-CoV-2-3CLpro (Table 1). Several old drugs such as Auranofin are newly discovered SARS-CoV-2-3CLpro inhibitors derived from old drugs, with IC50 reaching the single-digit micromolar level. The IC50 of the best compounds Auranofin, rabeprazole sodium, heparin sodium, disulfiram, ilaprazole, boceprevir and tenoprazole are 1.1μM, 1.5μM, 1.5μM, 1.6μM, 2.6μM, 4.8μM and 6.5μM, respectively (Figures 1-7).
由于各种冠状病毒中3CLpro及其底物结合口袋的高度保守性，这些新发现的抑制剂有望抑制其他冠状病毒3CLpro而发挥广谱的抗病毒活性。Due to the high conservation of 3CLpro and its substrate binding pocket among various coronaviruses, these newly discovered inhibitors are expected to inhibit other coronavirus 3CLpro and exert broad-spectrum antiviral activity.
实施例3：化合物2019新型冠状病毒复制抑制活性评价Example 3: Evaluation of the 2019-nCoV replication inhibitory activity of compounds
测定活性化合物(A1～A73)对各2019新型冠状病毒(SARS-CoV-2)复制抑制活性：将细胞在密度为5×104细胞/孔的48孔细胞培养皿中培养过夜，用不同浓度的活性化合物(金诺芬等多个老药)预先处理细胞1小时，然后加入病毒(多重感染复数MOI为0.05)使其感染2小时，然后取出病毒化合物混合物，用含活性化合物的新鲜培养基进一步培养细胞。在48h p.i.时，收集细胞上清液并在裂解缓冲液中裂解，通过定量实时RT-PCR(qRT-PCR)对细胞上清液中的病毒拷贝数进行定量评估，计算化合物抑制病毒的EC50。Determination of the inhibitory activity of active compounds (A1 to A73) on the replication of each 2019 novel coronavirus (SARS-CoV-2): The cells were cultured overnight in a 48-well cell culture dish at a density of 5×104 cells/well, and the cells were pre-treated with different concentrations of active compounds (multiple old drugs such as auranofin) for 1 hour, and then the virus (multiple infection MOI of 0.05) was added to infect for 2 hours, and then the virus compound mixture was removed and the cells were further cultured with fresh medium containing active compounds. At 48h pi, the cell supernatant was collected and lysed in lysis buffer, and the number of viral copies in the cell supernatant was quantitatively evaluated by quantitative real-time RT-PCR (qRT-PCR), and the EC50 of the compound's inhibition of the virus was calculated.
结果表明，本发明的活性化合物(金诺芬等多个老药)均可有效地抑制2019新型冠状病毒的复制，对不同分离的病毒株有一定的抑制。The results show that the active compounds of the present invention (many old drugs such as Auranofin) can effectively inhibit the replication of the 2019 novel coronavirus and have a certain degree of inhibition on different isolated virus strains.
讨论discuss
SARS-CoV-2 3CLpro与SARS-CoV 3CLpro的序列等同性高达96％，而底物结合口袋部分更是100％保守。2003年SARS-CoV爆发后针对SARS-CoV 3CLpro设计和发现了多个抑制剂，其多为共价作用的类肽和多肽抑制剂，同时有少量杂环酯类、吡唑类及大环类的非共价抑制剂见报道。这些已知抑制剂的生物活性主要通过体外酶水平检测，少量抑制剂在细胞水平具有一定的抑制效率(EC50多为微摩尔级别)。The sequence identity between SARS-CoV-2 3CLpro and SARS-CoV 3CLpro is as high as 96%, and the substrate binding pocket is 100% conserved. After the outbreak of SARS-CoV in 2003, several inhibitors were designed and discovered for SARS-CoV 3CLpro, most of which were covalent peptidomimetics and polypeptide inhibitors, and a small number of heterocyclic esters, pyrazoles and macrocyclic non-covalent inhibitors were reported. The biological activity of these known inhibitors is mainly detected at the in vitro enzyme level, and a small number of inhibitors have a certain inhibitory efficiency at the cellular level (EC50 is mostly at the micromolar level).
从已经批准上市的老药中寻找具有抗SARS-CoV-2活性的化合物是当前快速找到用于临床治疗COVID-19的最佳策略。Searching for compounds with anti-SARS-CoV-2 activity from old drugs that have been approved for marketing is currently the best strategy to quickly find clinical treatments for COVID-19.
在本发明之前，金诺芬等多个老药对冠状病毒3CL水解酶的抑制作用尚未见报道。Prior to the present invention, the inhibitory effects of several old drugs such as auranofin on coronavirus 3CL hydrolase have not been reported.
在本发明中，本发明人首次公开了金诺芬等多个老药对SARS-CoV-2 3CLpro具有显著的抑制作用(表1)，为老药来源的SARS-CoV-2 3CLpro新型抑制剂，IC50达到个位数微摩尔级别。由于各种冠状病毒中3CLpro及其底物结合口袋的高度保守性，本发明的SARS-CoV-2 3CLpro抑制剂有望抑制其他冠状病毒3CLpro而发挥广谱的抗病毒活性。In the present invention, the inventors disclosed for the first time that auranofin and other old drugs have significant inhibitory effects on SARS-CoV-2 3CLpro (Table 1), which are new SARS-CoV-2 3CLpro inhibitors derived from old drugs, with IC50 reaching single-digit micromolar levels. Due to the high conservation of 3CLpro and its substrate binding pocket in various coronaviruses, the SARS-CoV-2 3CLpro inhibitors of the present invention are expected to inhibit other coronavirus 3CLpro and exert broad-spectrum antiviral activity.
因此，金诺芬等多个老药为广谱抗单正链RNA病毒药物研发提供了重要的候选药物，具有良好的临床应用前景。Therefore, several old drugs such as Auranofin provide important candidate drugs for the development of broad-spectrum anti-single positive-strand RNA virus drugs and have good prospects for clinical application.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.
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| CN202310415534.XACN116236580B (en) | 2020-02-21 | 2020-02-21 | Application of old drugs such as Auranofin and their combination in combating single positive strand RNA viruses |
| Application Number | Priority Date | Filing Date | Title |
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| CN202310415534.XACN116236580B (en) | 2020-02-21 | 2020-02-21 | Application of old drugs such as Auranofin and their combination in combating single positive strand RNA viruses |
| CN202010109153.5ACN113289018B (en) | 2020-02-21 | 2020-02-21 | Application of old drugs such as auranofin and its composition in anti-single positive-strand RNA virus |
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| CN202010109153.5ADivisionCN113289018B (en) | 2020-02-21 | 2020-02-21 | Application of old drugs such as auranofin and its composition in anti-single positive-strand RNA virus |
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| CN116236580Btrue CN116236580B (en) | 2024-05-24 |
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| CN202310418461.XAPendingCN117427085A (en) | 2020-02-21 | 2020-02-21 | Application of old medicines such as auranofin and the like and compositions thereof in resisting single positive strand RNA viruses |
| CN202010109153.5AActiveCN113289018B (en) | 2020-02-21 | 2020-02-21 | Application of old drugs such as auranofin and its composition in anti-single positive-strand RNA virus |
| CN202310415534.XAActiveCN116236580B (en) | 2020-02-21 | 2020-02-21 | Application of old drugs such as Auranofin and their combination in combating single positive strand RNA viruses |
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| CN202310418461.XAPendingCN117427085A (en) | 2020-02-21 | 2020-02-21 | Application of old medicines such as auranofin and the like and compositions thereof in resisting single positive strand RNA viruses |
| CN202010109153.5AActiveCN113289018B (en) | 2020-02-21 | 2020-02-21 | Application of old drugs such as auranofin and its composition in anti-single positive-strand RNA virus |
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